Inhibitory effects of polysaccharides from Korean ginseng berries on LPS-induced RAW264.7 macrophages

Polysaccharides isolated from Korean ginseng berries (GBPs) have shown beneficial effects such as immunomodulatory, anti-inflammatory, anti-cancer, and anti-diabetic properties. However, little is known about anti-inflammatory effects of GBPs. Thus, the purpose of this study was to investigate anti-inflammatory properties of four fractions of GBPs, namely GBP-C, GBP-F1, GBP-F2, and GBP-F3, in macrophages. Their toxicities and effects on NO production in RAW264.7 cells were assessed by culturing cells with various concentrations of GBPs and stimulating cells with LPS. Furthermore, expression levels of inflammatory mediators, cytokines, cell surface molecules, and immune signaling pathways were evaluated in LPS-stimulated macrophages using different fractions of GBPs at 450 μg/mL. These GBPs activated LPS-stimulated RAW264.7 cells to significantly reduce NO production. They suppressed the expression of mRNA and cell surface molecules via MAPK and NF-κB pathways. Collectively, results revealed that all four GBP fractions showed anti-inflammatory effects, with GBP-F1 having a more efficient anti-inflammatory effect than GBP-C, GBP-F2, and GBP-F3. The structure of GBP-F1 mainly consists of 1 → 3)- Araf, 1 → 4)- Glcp, and 1 → 6)–Galp glycosidic linkages. These results demonstrate that GBPs can be employed as alternative natural sources of anti-inflammatory agents.


Introduction
Inflammation is a highly complex defense mechanism against outside harm or tissue damage [1].A variety of stimuli can cause inflammation, including pathogens, noxious chemical and mechanical agents, and autoimmune reactions [2].Macrophages are immune cells that play an important role in immunity.They can trigger an effective immune response by directly counteracting harmful stimuli such as lipopolysaccharide (LPS), muramyldipeptide (MDP), interferon-γ (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) [2,3].In inflammation, major functions of macrophages are to present antigen, to phagocytose, and to modulate immunity through various cytokines and growth factors [2].Additionally, macrophages could act as antigen-presenting cells (APCs) and modulate adaptive immunity by interacting with T lymphocytes [4].It has been found that macrophages can modulate LPS uptake and signaling following activation by releasing accessory molecules like cluster of differentiation (CD) 14 and toll-like receptors 4 (TLR4) [6,7].Macrophages can also produce co-stimulatory molecules such as CD40, CD80, and CD86 that can promote sustained stimulatory interactions of T cells [5,6].
A previous study has identified crude and three fractionated polysaccharides isolated from Korean ginseng berries (GBPs: GBP-C, GBP-F1, GBP-F2, and GBP-F3) by an ion-exchange chromatography.In the analysis of these polysaccharides, gas chromatography-mass spectrometry (GC-MS) has been used to evaluate monosaccharides such as rhamnose, arabinose, mannose, glucose, and galactose [32].Four fractions of polysaccharides have been shown to possess immune-enhancing activities by increasing the expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6 via NF-κB and MAPK signaling pathways [32].GBP-C can also boost immune function in mice [33].However, GBPs have not been explored for their anti-inflammatory properties.Thus, the objective of the current study was to determine anti-inflammatory effects of GBPs in RAW246.7 cells treated with LPS and probable mechanisms involved in such effects.
GBPs were diluted in RPMI-1640 medium (Gibco™, USA) supplemented with 1% FBS and 1% PS to different concentrations (50, 100, 250, and 450 μg/mL).For treatment groups, GBPs were added to cells at different concentrations.The negative control group was incubated with RPMI medium only.The positive control group was incubated with LPS only at a final concentration of 1 μg/mL.After 1 h of incubation, LPS at a final concentration of 1 μg/mL (Sigma-Aldrich, USA) was added to cells in groups treated with GBPs followed by incubation for another 24 h before performing assays to determine inhibitory effects of GBPs on inflammation.

Determination of cytotoxicity and NO production
The culture medium of treated cells was collected after LPS stimulation to measure NO generation using Griess reagent (Promega, USA).Briefly, 100 μL of the culture medium was mixed with 50 μL of Griess reagent A (1% sulfanilamide in 5% phosphoric acid) and incubated at room temperature for 5 min.After that, 50 μL of Griess reagent B (0.1% N-1-napthylethylenediamine dihydrochloride in water) was added followed by incubation for another 5 min.The absorbance of the solution was measured at 540 nm using a microplate reader (EL-800; BioTek Instruments, USA).An EZ-Cytox Cell Viability Assay Kit (Daeil Lab Service, Korea) was used to test the cytotoxicity of GBPs.The supernatant was discarded.The EZ-Cytox reagent was added to cells followed by incubation at 37˚C for 1 h.Absorbance at 450 nm was then measured using a microplate reader.

Real-time PCR analysis
TRI reagent1 (Molecular Research Center Inc., USA) was used to extract total RNA from cells.Reverse transcription of RNA to cDNA was performed with a High-capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA).Real-time PCR was carried out to determine expression levels of genes (namely iNOS, COX-2, IL-1β, IL-6, TNF-α, and β-actin) using TB Green1 Premix Ex Taq™ II (Takara Bio Inc., Japan) with a QuantStudio™ 3 FlexReal-Time PCR System (Thermo Fisher Scientific, USA).Primer sequences are listed in Table 1.

Western blot analysis
After treatments, cells were lysed with RIPA buffer (Tech & Innovation, China) for 30 min before centrifuging at 12,000×g for 10 min at 4˚C to prepare cell lysates containing proteins.These cell lysates were used to measure protein concentrations with a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, USA).SDS-PAGE and western blotting were then performed.Primary antibodies against p-NF-κB p65, p-p38, p-JNK, p-ERK1/2 (Cell Signaling Technology, USA) and α-tubulin (Abcam, UK) were used to immunoblot proteins, followed by incubation with goat anti-rabbit IgG(H+L)-HRP (GenDEPOT, USA).Detection of protein signals was conducted using Pierce 1 ECL Plus Western Blotting Substrate (Thermo Fisher Scientific, USA).A ChemiDoc XRS+ imaging system (Bio-Rad, USA) was then used to capture protein bands.

Flow cytometry assay
Accessory/ co-stimulatory molecules of cell-surface expression of CD14, CD40, CD86, and TLR4 were identified using flow cytometry.Cells were harvested and washed in a cold-buffer containing 2% FBS and 0.1% sodium azide in phosphate-buffered saline (PBS) before blocking with 50 μL of purified rat IgG (eBioscience Inc., USA) for 10 min.Antibodies against CD40-PE and CD86-APC (eBioscience Inc., USA) were added to the same tubes of cells containing Rat IgG2a kappa-PE and Rat IgG2a kappa-APC, respectively.Antibodies against CD14-FITC and TLR4-PE (eBioscience Inc., USA) were added to the same tubes of cells containing Rat IgG2a kappa-FITC and Rat IgG2a kappa-PE, respectively.All tubes of cells were incubated at 4˚C for an additional 20 min.These cells were rinsed twice with FACS buffer before recording flow cytometry data with a CytoFLEX Flow Cytometer (Beckman Coulter Inc., USA).

Glycosidic linkage and nuclear magnetic resonance (NMR) analysis
GBP-F1 was analyzed for glycosidic linkage using the approach previously established by Ciucanu and Kerek [39].A GC-MS system (6890 N/MSD 5973) equipped with an HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) (Agilent Technologies, USA) was used for the analysis of glycosidic linkage as described previously [40].

Gene
Forward primers Reverse primers To analyze the 1 H NMR spectrum, the most immunobiological polysaccharide GBP-F1 was dissolved in D 2 O. NMR spectra were generated using a JEOL ECA-600 spectrometer (JEOL, Akishima, Japan) coupled with a 5 mm multinuclear auto-turning TH tunable probe at a base frequency (600 MHz).

Data analysis
All data were analyzed using IBM SPSS Statistics version 23.0 (SPSS Inc., Chicago, IL, USA).The mean ± standard deviation (SD) of an independent study of three replicates was calculated.Results were examined using one-way analysis of variance (ANOVA) along with Duncan's multiple range tests.A value of p < 0.05 was regarded as statistically significant.

Cytotoxicity of GBPs to LPS-stimulated RAW264.7 cells
Fig 1 shows a significant increase in cell viability after treatment with LPS alone compared to the control (100%).GBP-C, GBP-F2, and GBP-F3 were not cytotoxic to macrophages at 450 μg/mL.GBP-F1 was not cytotoxic to RAW264.7 cells at concentrations below 250 μg/mL, although it was slightly cytotoxic (92%) at 450 μg/mL.Likewise, ginsenosides isolated from ginseng berries were not cytotoxic to RAW264.7 cells at concentrations between 62.5 and 500 μg/mL [21].These results revealed that GBPs had no cytotoxicity at concentrations up to 450 μg/mL.

GBPs suppress secretion of inflammatory mediators
Since the production of PGE 2 and NO is a significant indicator of inflammation [11,17,41], we evaluated effects of GBPs on their production after macrophages were stimulated with LPS.The release of NO production by GBPs in LPS-stimulated RAW246.7 cells was quantified with the Griess reaction method.According to our findings, there were significant dose-related reductions in NO production (50-450 μg/mL) with all GBPs compared to LPS (Fig 2).When LPS-stimulated RAW264.7 cells were treated with GBP-F1 at 450 μg/mL, the highest suppressive effect on NO production was 55%.For subsequent experiments, a concentration of 450 μg/mL was selected for GBPs.
The production of PGE 2 was also examined.Prior to stimulation with LPS, cells were pretreated with GBPs at 450 μg/mL.PGE 2 concentrations secreted by macrophage cells were then evaluated using an ELISA kit after 24 h of treatment.As shown in Fig 3A, LPS alone considerably boosted PGE 2 production in comparison with RPMI, whereas GBPs inhibited levels of PGE 2 production similar to those observed with NO production.GBPs also down-regulated the expression of inflammatory mediators such as iNOS and COX-2 when compared to LPS (Fig 3B and 3C).These findings indicated that GBPs suppressed NO and PGE 2 production via transcriptional regulation of iNOS and COX-2, respectively.

GBPs suppress expression of cytokines
In order to assess effects of GBPs on secretion levels of pro-inflammatory cytokines by LPSstimulated macrophages, real-time PCR assay and ELISA kits were used to analyze mRNA and protein levels, respectively.GBPs reduced levels of IL-1β, IL-6, and TNF-α in RAW264.7 cells stimulated with LPS (Fig 4A -4C).As shown in Fig 4D -4F, similar results were obtained for protein levels of IL-1β, IL-6, and TNF-α.Thus, GBPs inhibited LPS-induced release of inflammatory cytokines, with GBP-F1 having the highest anti-inflammatory effects.These findings demonstrate that all GBPs, particularly GBP-F1, have potential anti-inflammatory effects.

GBPs inhibit surface molecule expression
As shown in Fig 6A and 6B, LPS dramatically up-regulated the expression of CD14 and TLR4.However, cell surface expression was considerably decreased by GBPs when compared with LPS alone.GBPs decreased the expression of CD14 and TLR4 in LPS-stimulated macrophages.These findings indicated that GBPs could attenuate LPS binding to the surface of RAW264.7 cells.
In order to examine surface biomarkers of RAW264.7 cells, CD40 and CD86 were subjected to flow cytometry analysis.GBPs were studied for their anti-inflammatory effects on CD40 and CD86 expression.Mean fluorescence intensities (MFI) are shown in Fig 6 .GBPs decreased the expression of CD86 but not the expression of CD40 on the surface of LPS-stimulated macrophages (Fig 6C and 6D).
Glycosidic linkages of GBP-F1 polymer were further identified from 1D NMR spectra (Fig 7 ) with 1 H analysis to support the ten derivatives of glycosidic linkages residues.All ten anomeric signals were noticed from 1

Discussion
In our preliminary research study, we have isolated, purified, and characterized a crude polysaccharide and fractionated polysaccharides (GBP-C, GBP-F1, GBP-F2, and GBP-F3) and further confirmed that these four polysaccharides consist of five monosaccharides, including rhamnose (4.0-15.9%),arabinose (10.7-26.8%),mannose (2.2-5.9%),glucose (26.7-34.6%),and galactose (24.5-46.3%)[32].These four polysaccharides have also been demonstrated to improve immunological function of macrophages.However, their anti-inflammatory effects have not been reported yet.In this study, anti-inflammatory effects of Korean ginseng berry polysaccharides on macrophages were investigated.The mechanism by which these four polysaccharides suppressed LPS-induced inflammatory responses was also determined.
Macrophages play an essential role in both particular and non-specific immunological reactions throughout the inflammatory process by generating a variety of inflammation-related bioactive chemicals, mediators, and cytokines, most notably NO, PGE 2 , TNF-α, and several specific interleukins [2,9].NO synthesized from arginine by nitric oxide synthase (NOS) is an important mediator of inflammation in the body since it is a neurotransmitter and a defense molecule against bacteria, parasites, and tumor cells [9].In the present study, results indicated that NO generation was decreased in groups treated with GBPs at 50-450 μg/mL.A high inhibition on NO production was found in the GBP-F1 group.These results might be due to expression of iNOS induced by GBPs in LPS-stimulated RAW264.7 cells.Saponins extracted from ginseng and sulfated polysaccharides from brown alga can also effectively suppress LPSinduced NO production up to 400 μg/mL of the concentration of saponin extracts [10,14].PGE 2 is produced at the inflammatory site by COX-2.It is also implicated as an important mediator in the process of inflammation.Thus, reducing iNOS and COX-2 expression in macrophages may be an efficient trigger for inhibiting the production of NO and PGE 2 to suppress inflammation.Our results showed that GBPs at 450 μg/mL also reduced PGE 2 production and mRNA expression levels of iNOS and COX-2.Similarly, guluronate oligosaccharides from alginate also decreased expression levels of mRNA and proteins of iNOS and COX-2 in LPS-stimulated macrophages [41].These findings suggest that GBPs can decrease NO and PEG 2 production by decreasing iNOS and COX-2 expression, thus exerting potent anti-inflammatory effects.Polysaccharides from Smilax glabra Roxb., such as SGP-1 and SGP-2, have been shown to be able to reduce iNOS, TNF-α , and IL-6 expression in LPS-stimulated macrophages [16].TNF-α, IL-6, and IL-1β are primary cytokines generated by activated macrophages during initial inflammatory responses of host defense and infection [2], consistent with previous reports [10,21].In this study, GBPs dramatically reduced the synthesis of IL-1β, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells, indicating that GBPs could inhibit macrophage immune responses by directly increasing cytokine production and mRNA expression.
Recently, numerous studies have discovered that polysaccharides produced from medicinal plants possess anti-inflammatory activities by blocking activation of NF-κB and MAPK signaling pathways in macrophages [16][17][18].Transcription factor NF-κB is important for regulating inflammation-induced enzymes and cytokines in a variety of cellular functions, including NF-κB1 (p50/p105), NF-κB2 (p52/p100), p65 (RelA), RelB, and c-Rel, which are recognized as biomarkers of NF-κB activation [42].Activated NF-κB p65 can migrate to the nucleus and regulate inflammation-related genes such as iNOS and pro-inflammatory cytokines [10].Interestingly, our results revealed that GBPs reduced NF-κB release in LPS-stimulated RAW264.7 cells by reducing NF-κB phosphorylation and translocation to the nucleus.The MAPK pathway is also a key intracellular signaling cascade in immune responses.MAPK has three families, namely, p38 MAPK, JNK/stress activated protein kinase, and ERK [43].Our results also demonstrated that GBPs suppressed ERK1/2 and p38 phosphorylation.These findings imply that anti-inflammatory actions of GBPs via NF-κB signaling regulation might contribute to their inhibitory effects on MAPK activity.
LPS can stimulate macrophages to release inflammatory mediators related to TLR4 and CD14 molecules on the cell surface that play important roles in the immune system [44].LPS signaling appears when LPS is transferred from membrane to soluble CD14 by LPS binding protein, which is subsequently recognized by TLR4 [5,13].Guluronate oligosaccharides from alginate can decrease LPS-stimulated expression of TLR4 and CD14 molecules [41].Polysaccharides from thunder god vine (Tripterygium wilfordii Hook.f.) can also reduce the release of adhesion molecules including CD11c, CD18, CD14, and CD54 in THP-1 cells [45].Co-stimulatory molecules such as CD40 and CD86 are expressed on APCs and T cells, indicating active macrophages on their surfaces [5,11,18].In the present study, results indicated that CD86 expression decreased the immunological activity in RAW264.7 cells treated with GBPs.Reduced expression of CD40 and CD86 can alleviate diseases such as chronic inflammatory disease and autoimmune disease because they are crucial for productive interactions between T cells and APCs cells [5].These results suggest that surface molecules of CD14, CD86, and TLR4 might contribute to inflammatory responses of LPS-stimulated RAW264.7 macrophages treated by GBPs.

Conclusions
In summary, anti-inflammatory effects of all GBPs, including fractionated GBP-C, GBP-F1, GBP-F2, and GBP-F3, are mediated by inhibiting inflammatory mediators and cytokines as well as surface accessory/co-stimulatory molecules via activation of NF-κB and MAPK signaling pathways in LPS-stimulated RAW264.7 cells.In particular, GBP-F1 showed more potent anti-inflammation than GBP-C, GBP-F2, and GBP-F3 fractions.GBP-F1 demonstrated significant anti-inflammatory properties.It has the potential to be a natural product used in the development of therapeutic agents.

Fig 1 .
Fig 1. Cytotoxic effects of GBPs on RAW264.7 cells stimulated by LPS.Cells were treated with GBPs at various concentrations and stimulated with LPS.Cell viability was assessed using a WST test.Data from three separate experiments performed in duplicate are reported as means ± SD. *p < 0.05 vs. RPMI.https://doi.org/10.1371/journal.pone.0294675.g001

Fig 2 .
Fig 2. Effects of GBPs on LPS-stimulated NO production.Cells were treated with GBPs at various concentrations and stimulated with LPS.The amount of NO was determined with a Griess reagent.Data from three separate experiments performed in duplicate are reported as means ± SD. *p < 0.05 vs. LPS.https://doi.org/10.1371/journal.pone.0294675.g002

Fig 5
Fig 5 shows the anti-inflammatory effects of GBPs on NF-κB and MAPK activation and immune-blotting results.The phosphorylation of NF-κB-p65 was decreased by GBPs in comparison to LPS alone, whereas the phosphorylation of NF-κB-p65 was significantly elevated by GBPs in comparison to the negative control (RPMI).GBPs were also observed in the phosphorylation of ERK, JNK, and p38 in the same pattern of NF-κB activation (Fig 5).Interestingly, GBP-F1 inhibited the phosphorylated NF-κB-p65, ERK, JNK, and p38 in LPSstimulated RAW264.7 cells at the highest level among the other GBPs.These findings demonstrated that all GBPs, particularly GBP-F1, could suppress the activation of the NF-κB and MAPK pathways.

Fig 3 .Fig 4 .
Fig 3. Effects of GBPs on LPS-stimulated PGE 2 production and inflammatory mediators.Cells were treated with various polysaccharides at 450 μg/mL and stimulated with LPS at 1 μg/mL.Amounts of PGE 2 (A) were assessed using ELISA kits.The relative expression of iNOS (B) and COX-2 (C) was assessed using real-time PCR.Data are presented as means ± SD.A different letter (p < 0.05) indicates statistically significant differences between treatments.https://doi.org/10.1371/journal.pone.0294675.g003

Fig 5 .
Fig 5. Effects of GBPs on protein expression by suppressing immune signaling pathways.Cells were treated with various GBPs at 450 μg/mL and stimulated with LPS at 1 μg/mL.Effects of GBPs on protein expression of NF-κB and MAPK pathways were determined by western blotting.Data from three separate experiments performed in duplicate are reported as means ± SD.A different letter (p < 0.05) indicates statistically significant differences between treatments.https://doi.org/10.1371/journal.pone.0294675.g005